Frederick A. Villamena

Detection of Reactive Oxygen and Nitrogen Species by EPR Spin Trapping

Electron paramagnetic resonance spin trapping has become an indispensable tool for the specific detection of reactive oxygen free radicals in biological systems. In this review we describe some of the advantages as well as some experimental considerations of this technique and how it can be applied to biological systems to measure oxidative stress.

Acute DPP-4 inhibition modulates vascular tone through GLP-1 independent pathways ☆

Evidence from both clinical and experimental studies indicates that Di-peptidyl peptidase-IV (DPP-4) inhibition may mediate favorable effects on the cardiovascular system. The objective of this study was to examine the acute effects of DPP-4 inhibition on vascular responses and to study the underlying mechanisms of alteration in tone. Aortic segments from C57BL/6 mice were treated with vasoconstrictors and exposed to various doses of alogliptin, a selective DPP-4 inhibitor. Vasodilator responses were evaluated using pathway specific antagonists to elucidate mechanisms of response. In parallel experiments, cultured human umbilical vein endothelial cells (HUVEC) were exposed to varying concentrations of alogliptin to evaluate the effects on candidate vasodilator pathways. Alogliptin relaxed phenylephrine and U46619 pre-constricted aortic segments in a dose dependent manner. Relaxation responses were not affected by the glucagon-like peptide-1 (GLP-1) receptor antagonist, exendin fragment 9-39 (88 ± 6 vs. 91 ± 2, p < 0.001). Vascular relaxation to alogliptin was significantly decreased by endothelial denudation, L-N(G)-monomethyl-arginine citrate (L-NMMA) and by the soluble guanylate cyclase inhibitor ODQ. DPP-4 inhibition induced relaxation was completely abolished by a combination of L-NMMA, charybdotoxin and apamin. Incubation of HUVECs with alogliptin resulted in eNOS and Akt phosphorylation (Ser(1177) and Ser(473) respectively) paralleled by a rapid increase in nitric oxide. Inhibition of Src kinase decreased eNOS and Akt phosphorylation, in contrast to a lack of any effect on insulin mediated activation of the eNOS-Akt, suggesting that alogliptin mediates vasodilation through Src kinase mediated effects on eNOS-Akt. DPP-4 inhibition by alogliptin mediates rapid vascular relaxation via GLP-1 independent, Src-Akt-eNOS mediated NO release and the activation of vascular potassium channels.

Synthesis and Electrochemical and Biological Studies of Novel Coumarin–Chalcone Hybrid Compounds

A series of novel hydroxy-coumarin-chalcone hybrid compounds 2a-i has been synthesized by employing a simple and efficient methodology. An electrochemical characterization using cyclic voltammetry and ESR spectroscopy were carried out to characterize the oxidation mechanism for the target compounds. The antioxidant capacity and reactivity were determined by ORAC and ESR assays, respectively. Biological assays were assessed to evaluate the cytotoxicity and cytoprotection capacity against ROS/RNS on BAEC. The results revealed that all tested compounds present ORAC values that are much higher than other well-known antioxidant compounds such as quercetin and catechin. Compound 2e showed the highest ORAC value (14.1) and also presented a low oxidation potential, good scavenging capacity against hydroxyl radicals, low cytotoxicity, and high cytoprotective activity.

CD36-Dependent 7-Ketocholesterol Accumulation in Macrophages Mediates Progression of Atherosclerosis in Response to Chronic Air Pollution Exposure

Rationale: Air pollution exposure has been shown to potentiate plaque progression in humans and animals. Our previous studies have suggested a role for oxidized lipids in mediating adverse vascular effect of air pollution. However, the types of oxidized lipids formed in response to air pollutants and how this occurs and their relevance to atherosclerosis are not fully understood. Objective: To investigate the mechanisms by which particulate matter <2.5 &mgr;m (PM2.5) induces progression of atherosclerosis. Methods and Results: Atherosclerosis-prone ApoE−/− or LDLR−/− mice were exposed to filtered air or concentrated ambient PM2.5 using a versatile aerosol concentrator enrichment system for 6 months. PM2.5 increased 7-ketocholesterol (7-KCh), an oxidatively modified form of cholesterol, in plasma intermediate density lipoprotein/low-density lipoprotein fraction and in aortic plaque concomitant with progression of atherosclerosis and increased CD36 expression in plaque macrophages from PM2.5-exposed mice. Macrophages isolated from PM2.5-exposed mice displayed increased uptake of oxidized lipids without alterations in their efflux capacity. Consistent with these finding, CD36-positive macrophages displayed a heightened capacity for oxidized lipid uptake. Deficiency of CD36 on hematopoietic cells diminished the effect of air pollution on 7-KCh accumulation, foam cell formation, and atherosclerosis. Conclusions: Our results suggest a potential role for CD36-mediated abnormal accumulations of oxidized lipids, such as 7-KCh, in air pollution–induced atherosclerosis progression.

Lipoic acid effects on established atherosclerosis

AIMS Alpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis. MAIN METHODS Watanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-kappaB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA). KEY FINDINGS LA decreased body weight by 15+/-5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-kappaB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy. SIGNIFICANCE The present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.

Synthesis and Characterization of Ester-Derivatized Tetrathiatriarylmethyl Radicals as Intracellular Oxygen Probes

Electron paramagnetic resonance (EPR) spectroscopy using paramagnetic probes has been employed as an important tool for the accurate determination of oxygen (O2) concentrations in biological systems. However, paramagnetic probes are still limited by their intracellular penetrability. Various esterified trityl derivatives were synthesized and characterized, and an X-ray structure of one of the triyl radicals was determined. The ester-derivatized trityls exhibited higher sensitivity to O2 concentration compared to the trityl tricarboxylate CT-03. Cyclic voltammetry was also carried out to assess the susceptibility of the trityl radicals to oxidation and reduction. Among all of the ester-derivatized trityls studied, facile hydrolysis of the acetoxymethoxy esters to the respective carboxylate was observed using porcine liver esterase. This study demonstrates that cellular permeability of the trityl radicals can be achieved by varying the type and number of ester groups. Therefore, ester-derivatized trityl radicals show great potential as intracellular EPR oximetry probes and imaging agents.

The Radical Trap 5,5-Dimethyl-1-Pyrroline N-Oxide Exerts Dose-Dependent Protection against Myocardial Ischemia-Reperfusion Injury through Preservation of Mitochondrial Electron Transport

Free radicals are important mediators of myocardial ischemia-reperfusion injury. Nitrone spin traps have been shown to scavenge free radicals. The cardioprotective effect of the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was investigated in an isolated heart model of global ischemia and reperfusion. Rat hearts were perfused and subjected to global ischemia for 30 min followed by reperfusion with four treatment groups of varying DMPO concentration (0.5-10 mM) administered before induction of ischemia. DMPO treatment improved the recovery of left ventricular (LV) function and coronary flow over the 30-min period of reperfusion compared with untreated hearts. Enhanced recovery was observed for all doses studied but was highest with 1 mM treatment with 2.4-fold higher recovery of LV developed pressure and 37% reduction in infarct size. Superoxide was measured by tissue fluorometry using the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} probe hydroethidine. Hearts treated with 1 mM DMPO showed a significant reduction in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} production compared with control hearts both over the first 5 min of ischemia and upon reperfusion after 30 min of global ischemia. Studies of mitochondrial function demonstrated that 1 mM DMPO increased the recovery of function of complexes I, II/III, and IV after 30 min of reperfusion. Immunoblotting with antibodies against complexes I, II, and IV further revealed marked up-regulation of mitochondrial proteins, suggesting that DMPO prevents their ischemic degradation via scavenging oxygen radicals generated during ischemia/reperfusion. Thus, DMPO functions as a protective agent against ischemic and postischemic injury via radical scavenging, conferring robust dose-dependent protection with salvage of mitochondrial function and redox homeostasis.

Improved Spin Trapping Properties by β-Cyclodextrin−Cyclic Nitrone Conjugate

Spin trapping using a nitrone and electron paramagnetic resonance (EPR) spectroscopy is commonly employed in the identification of transient radicals in chemical and biological systems. There has also been a growing interest in the pharmacological activity of nitrones, and there is, therefore, a pressing need to develop nitrones with improved spin trapping properties and controlled delivery in cellular systems. The beta-cyclodextrin (beta-CD)-cyclic nitrone conjugate, 5-N-beta-cyclodextrin-carboxamide-5-methyl-1-pyrroline N-oxide (CDNMPO) was synthesized and characterized. 1-D and 2-D NMR show two stereoisomeric forms (i.e., 5S- and 5R-) for CDNMPO. Spin trapping using CDNMPO shows distinctive EPR spectra for superoxide radical anion (O2(*-)) compared to other biologically relevant free radicals. Kinetic analysis of O2(*-) adduct formation and decay using singular value decomposition and pseudoinverse deconvolution methods gave an average bimolecular rate constant of k = 58 +/- 1 M(-1) s(-1) and a maximum half-life of t(1/2) = 27.5 min at pH 7.0. Molecular modeling was used to rationalize the long-range coupling between the nitrone and the beta-CD, as well as the stability of the O2(*-) adducts. This study demonstrates how a computational approach can aid in the design of spin traps with a relatively high rate of reactivity to O2(*-), and how beta-CD can improve adduct stability via intramolecular interaction with the O2(*-) adduct.

Theoretical and experimental studies of the spin trapping of inorganic radicals by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 3. Sulfur dioxide, sulfite, and sulfate radical anions.

Radical forms of sulfur dioxide (SO(2)), sulfite (SO(3)(2-)), sulfate (SO(4)(2-)), and their conjugate acids are known to be generated in vivo through various chemical and biochemical pathways. Oxides of sulfur are environmentally pervasive compounds and are associated with a number of health problems. There is growing evidence that their toxicity may be mediated by their radical forms. Electron paramagnetic resonance (EPR) spin trapping using the commonly used spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been employed in the detection of SO(3)(•-) and SO(4)(•-). The thermochemistries of SO(2)(•-), SO(3)(•-), SO(4)(•-), and their respective conjugate acids addition to DMPO were predicted using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level. No spin adduct was observed for SO(2)(•-) by EPR, but an S-centered adduct was observed for SO(3)(•-)and an O-centered adduct for SO(4)(•-). Determination of adducts as S- or O-centered was made via comparison based on qualitative trends of experimental hfccs with theoretical values. The thermodynamics of the nonradical addition of SO(3)(2-) and HSO(3)(-) to DMPO followed by conversion to the corresponding radical adduct via the Forrester-Hepburn mechanism was also calculated. Adduct acidities and decomposition pathways were investigated as well, including an EPR experiment using H(2)(17)O to determine the site of hydrolysis of O-centered adducts. The mode of radical addition to DMPO is predicted to be governed by several factors, including spin population density, and geometries stabilized by hydrogen bonds. The thermodynamic data supports evidence for the radical addition pathway over the nucleophilic addition mechanism.

Esterified Trityl Radicals as Intracellular Oxygen Probes

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O2 concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O2 concentrations and the effects of menadione and KCN on the rates of O2 consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O2 concentration and consumption.